Method for extracting DNA from organisms

Abstract

A method for the indirect extraction of DNA greater than 300 kb in size from non-cultivatable organisms contained in an environmental sample is disclosed. The method comprises isolating the organisms from the sample and embedding the isolated organisms in a block of agarose where the organisms are subsequently lysed and the DNA subjected to a first alternating field electrophoresis to extract DNA fragments which are large in size and separate them from the cell debris. The first electrophoretic migration is followed by an enzymatic restriction step and additional electrophoretic migrations. The invention also encompasses DNA obtained by the method.

1 . A method for extracting DNA from non-cultivatable organisms contained in a sample, said method comprising: 
 (a) grinding said sample in liquid medium;    (b) recovering the ground material obtained in (a);    (c) isolating said organisms from the ground material by sedimentation using a density gradient solution with a density greater than 1;    (d) embedding the organisms isolated in (c) in a block of agarose;    (e) releasing DNA of said organisms by lysis of said organisms in the block of agarose;    (f) placing said block in a well of an agarose gel and subjecting said block to a first electrophoretic migration comprising exposure to at least one alternating field electrophoresis; and    (g) recovering DNA from the agarose gel.    
   
   
       2 - 13 . (canceled)  
   
   
       14 . DNA of non-cultivatable organisms obtained by the method of  claim 1 .  
   
   
       15 . The DNA of  claim 14 , wherein said DNA is greater than 300 kB in size.  
   
   
       16 . A DNA library consisting of DNA fragments obtained from the method of  claim 1 .  
   
   
       17 . Size-homogeneous DNA fragments obtained by the method of  claim 1 .  
   
   
       18 . Size-homogeneous DNA fragments obtained by the method of  claim 1  wherein said fragments are from environmental samples.  
   
   
       19 . Size-homogeneous DNA fragments greater than 300 kb in size obtained by the method of  claim 1 .  
   
   
       20 . Size-homogeneous DNA fragments greater than  300  kb in size obtained by the method of  claim 1  wherein said fragments are from environmental samples.  
   
   
       21 . A library of size-homogeneous DNA obtained by the method of  claim 1 .  
   
   
       22 . A library of size-homogeneous DNA obtained by the method of  claim 1  wherein said fragments are from environmental samples.  
   
   
       23 . A library of size-homogeneous DNA fragments greater than 300 kb in size obtained by the method of  claim 1 .  
   
   
       24 . A library of size-homogeneous DNA fragments greater than 300 kb in size obtained by the method of  claim 1  wherein said fragments are from environmental samples.